Evaluation of a monoclonal antibody-based immunoassay for detecting type A Clostridium botulinum toxin produced in pure culture and an inoculated model cured meat system

A monoclonal antibody-based amplified enzyme-linked immunosorbent assay (ELISA) method for detecting Clostridium botulinum type A toxin was evaluated for its ability to detect the toxin in the supernatant fluid of pure cultures and after growth from Cl. botulinum spores inoculated into pork slurries. Slurries containing NaCl (1.5-4.5% w/v) and polyphosphate (0.3% w/v) were either unheated or heated, 80 degrees C/5 min + 70 degrees C/2 h, before storage at 15 degrees, 20 degrees or 27 degrees C. The presence of specific toxin was confirmed by mouse bioassay and results compared with those of the amplified ELISA method. A total of 49 strains, 39 Cl. botulinum and 10 Cl. sporogenes (putrefactive anaerobes), and 95 slurry samples were tested. Fourteen of 15 strains of type A Cl. botulinum and 34 of 36 slurry samples containing type A toxin were positive by ELISA. No false positive reactions occurred with Cl. botulinum types B, C, D, E and F, or with the 10 strains of Cl. sporogenes. However, toxin produced by one strain of Cl. botulinum type A (NCTC 2012) was not detected by the amplified ELISA.     

Growth and formation of toxin by Clostridium botulinum in peeled, inoculated, vacuum-packed potatoes after a double pasteurization and storage at 25 degrees C

A process that claims to use a double pasteurization to produce vacuum-packed potatoes for storage at ambient temperature has been evaluated. After the first pasteurization, potatoes are vacuum-packed and stored at 25 degrees-35 degrees C for up to 24 h, which is intended to allow germination of bacterial spores, and are then pasteurized again. When potatoes were inoculated with spores of Clostridium botulinum and subjected to this double-pasteurization process a high proportion of spores remained viable and resulted in growth and formation of toxin within 5-9 d at 25 degrees C. To provide an appropriate reduction in the risk o survival and growth of Cl. botulinum, peeled, vacuum-packed potatoes for storage at ambient temperature should be given a heat treatment equivalent to an F(0)3 process. If they are not given such a heat treatment they should be stored at a temperature below 4 degrees C.     

Evaluation of a monoclonal antibody-based immunoassay for detecting type B Clostridium botulinum toxin produced in pure culture and an inoculated model cured meat system

A monoclonal antibody-based amplified ELISA method for detecting Clostridium botulinum type B toxin was evaluated for its ability to detect the toxin in the supernatant fluid of pure cultures and after growth from Cl. botulinum spores inoculated into pork slurries. Slurries containing NaCl (1.5-4.5% w/v) and polyphosphate (0.3% w/v) were either unheated or heated 80 degrees C/5 min followed by 70 degrees C/2 h before incubation at 15 degrees, 20 degrees or 27 degrees C. Presence of specific toxin was confirmed by mouse bioassay and results were compared with those of the amplified ELISA method. A total of 48 strains, consisting of 38 Cl. botulinum and 10 Cl. sporogenes (putrefactive anaerobes), and 140 slurry samples were tested. Cultures of eight out of nine strains of type B Cl botulinum and 73 of 101 slurry samples containing type B toxin were positive by ELISA; the remaining 28 slurry samples contained type B toxin at levels below or close to the detection limit (20 LD50/ml) of the type B ELISA. No false-positive reactions occurred with Cl. botulinum types A, C, D, E or F, or with the 10 strains of Cl. sporogenes. Toxin produced by one strain of Cl. botulinum type B (NCTC 3807) was not detected by this single monoclonal antibody-based amplified ELISA. With a mixture of two monoclonal antibodies, however, the toxin from NCTC 3807 could be detected without reducing the sensitivity of the ELISA.     

Evaluation of a monoclonal antibody-based immunoassay for detecting type A Clostridium botulinum toxin produced in pure culture and an inoculated model cured meat system

A monoclonal antibody-based amplified enzyme-linked immunosorbent assay (ELISA) method for detecting Clostridium botulinum type A toxin was evaluated for its ability to detect the toxin in the supernatant fluid of pure cultures and after growth from Cl. botulinum spores inoculated into pork slurries. Slurries containing NaCl (1.5–4.5% w/v) and polyphosphate (0.3% w/v) were either unheated or heated, 80°C/5 min + 70°C/2 h, before storage at 15°, 20° or 27°C. The presence of specific toxin was confirmed by mouse bioassay and results compared with those of the amplified ELISA method. A total of 49 strains, 39 Cl. botulinum and 10 Cl. sporogenes (putrefactive anaerobes), aiid 95 slurry samples were tested. Fourteen of 15 strains of type A Cl. botulinum and 34 of 36 slurry samples containing type A toxin were positive by ELISA. No false positive reactions occurred with Cl. botulinum types B, C, D, E and F, or with the 10 strains of Cl. sporogenes. However, toxin produced by one strain of Cl. botulinum type A (NCTC 2012) was not detected by the amplified ELISA.     

The abattoir source of culturable psychrophilic Clostridium spp. causing 'blown pack' spoilage of vacuum-packed chilled venison

AIMS: To identify the abattoir source(s) of culturable psychrophilic clostridia causing 'blown pack' spoilage of vacuum-packed chilled meats. METHODS AND RESULTS: Psychrophilic and psychrotolerant clostridia were isolated from hides, faeces and tonsils of deer slaughter stock, and from a meat plant environment. The isolates were differentiated using restriction fragment length polymorphism analysis of the 16S rDNA gene (PCR-RFLP) and 16S-23S rDNA internal transcribed spacer (ITS) analysis. PCR-RFLP group I clostridia were found to have restriction patterns indistinguishable from the patterns of 'blown pack'-causing Clostridium gasigenes DB1A(T) and R26. Gas production in packs inoculated with vegetative cells of PCR-RFLP group I clostridia was first evident after 14 days at 2 degrees C. The prevalence of these clostridia was similar in hide and faecal samples from slaughter animals, but these micro-organisms were absent from tonsils and the meat plant environment. Banding patterns of PCR-RFLP group II clostridia showed some cross-similarity with patterns of the 'blown pack'-causing micro-organism Cl. estertheticum DSM 8809(T) and Cl. estertheticum-like meat strains. The majority of clostridia in PCR-RFLP group II were found in the faeces of slaughter animals. Isolates representing PCR-RFLP group II did not, however, produce gas in vacuum packs stored at 2 degrees C for 84 days. CONCLUSIONS: The data suggest that soil particles attached to hide or present in faeces are the most probable primary reservoir from which 'blown pack' clostridia are introduced onto carcasses. Therefore, dressing procedure hygiene remains paramount in order to control the spread of psychrophilic Clostridium spp. in a meat plant. SIGNIFICANCE AND IMPACT OF THE STUDY: The paper provides information critical for controlling 'blown pack' spoilage in meat processing plants. It reports on the use of molecular techniques for determination of abattoir sources of 'blown pack'-causing clostridia.     

Survival Studies with Spores of Clostridium botulinum Type E in Pasteurized Meat of the Blue Crab Callinectes sapidus

Clostridium botulinum type E studies reported in this paper include the incidence of the organism in selected Chesapeake Bay areas, growth and toxin production in crabmeat homogenates, and the effect of pasteurization upon varying levels of spores in crabmeat. Type E spores were detected in 21 of 24 bottom mud samples taken at locations from which blue crabs were being harvested. Sterilized crabmeat homogenates inoculated with as little as five spores per 10 g became toxic after 8 days at 50 F, 2 days at 75 F, and 1 day at 85 F. Growth at 50 F and above was accompanied by gas production and a slightly sour odor. Growth and toxin production at 40 F required 55 days or longer and inocula of 10(3) spores or higher per 10 g of homogenate. At 40 F gas production was usually not apparent and no off odors could be detected. A recommended minimum pasteurization of 1 min at 185 F internal meat temperature reduced type E spore levels in inoculated packs of crabmeat from 10(8) spores per 100 g to 6 or less spores per 100 g, and the pasteurized meat remained nontoxic during 6 months of storage at 40 F.     

Dependence of Clostridium botulinum gas and protease production on culture conditions

Reports that Clostridium botulinum toxin can sometimes be detected in the absence of indicators of overt spoilage led to a systematic study of this phenomenon in a model system. Media with various combinations of pH (5.0 to 7.0) and glucose (0.0 to 1.0%) were inoculated with vegetative cells of C. botulinum 62A and incubated anaerobically at 35 degrees C. Although growth and toxin production occurred at all pH and glucose combinations, accumulation of gas was delayed or absent in media with low pH, low glucose levels, or both. Other proteolytic C. botulinum strains gave similar results. Trypsin activation was required to detect toxin in some low pH cultures. The trypsinization requirement correlated with low proteolytic activity in the cultures. Proteolytic activity of the strains examined was 5- to 500-fold lower in botulinal assay medium than in cooked meat medium. The results indicate that the absence of gas accumulation does not preclude the presence of botulinal toxin and that proteolytic cultures grown under adverse conditions may require trypsinization for the detection of toxin.     

Dependence of Clostridium botulinum gas and protease production on culture conditions.

Reports that Clostridium botulinum toxin can sometimes be detected in the absence of indicators of overt spoilage led to a systematic study of this phenomenon in a model system. Media with various combinations of pH (5.0 to 7.0) and glucose (0.0 to 1.0%) were inoculated with vegetative cells of C. botulinum 62A and incubated anaerobically at 35 degrees C. Although growth and toxin production occurred at all pH and glucose combinations, accumulation of gas was delayed or absent in media with low pH, low glucose levels, or both. Other proteolytic C. botulinum strains gave similar results. Trypsin activation was required to detect toxin in some low pH cultures. The trypsinization requirement correlated with low proteolytic activity in the cultures. Proteolytic activity of the strains examined was 5- to 500-fold lower in botulinal assay medium than in cooked meat medium. The results indicate that the absence of gas accumulation does not preclude the presence of botulinal toxin and that proteolytic cultures grown under adverse conditions may require trypsinization for the detection of toxin.     

Spoilage-related activity of Carnobacterium maltaromaticum strains in air-stored and vacuum-packed meat

One hundred three isolates of Carnobacterium spp. from raw meat were analyzed by random amplification of polymorphic DNA (RAPD) and PCR and were identified by 16S rRNA gene sequencing. Forty-five strains of Carnobacterium maltaromaticum were characterized for their growth capabilities at different temperatures, NaCl concentrations, and pH values and for in vitro lipolytic and proteolytic activities. Moreover, their spoilage potential in meat was investigated by analyzing the release of volatile organic compounds (VOCs) in meat stored in air or vacuum packs. Almost all the strains were able to grow at 4, 10, and 20°C, at pH values of 6 to 9, and in the presence of 2.5% NaCl. The release of VOCs by each strain in beef stored at 4°C in air and vacuum packs was evaluated by headspace solid-phase microextraction (HS-SPME)-gas chromatography-mass spectrometry (GC-MS) analysis. All the meat samples inoculated and stored in air showed higher numbers of VOCs than the vacuum-packed meat samples. Acetoin, 1-octen-3-ol, and butanoic acid were the compounds most frequently found under both storage conditions. The contaminated meat samples were evaluated by a sensory panel; the results indicated that for all sensory odors, no effect of strain was significant (P > 0.05). The storage conditions significantly affected (P < 0.05) the perception of dairy, spoiled-meat, and mozzarella cheese odors, which were more intense in meat stored in air than in vacuum packs but were never very intense. In conclusion, different strains of C. maltaromaticum can grow efficiently in meat stored at low temperatures both in air and in vacuum packs, producing volatile molecules with low sensory impacts, with a negligible contribution to meat spoilage overall.     

Growth and formation of toxin by Clostridium botulinum in peeled, inoculated, vacuum-packed potatoes after a double pasteurization and storage at 25°C

A process that claims to use a double pasteurization to produce vacuum-packed potatoes for storage at ambient temperature has been evaluated. After the first pasteurization, potatoes are vacuum-packed and stored at 25°C–35°C for up to 24 h, which is intended to allow germination of bacterial spores, and are then pasteurized again. When potatoes were inoculated with spores of Clostridium botulinum and subjected to this double-pasteurization process a high proportion of spores remained viable and resulted in growth and formation of toxin within 5–9 d at 25°C. To provide an appropriate reduction in the risk of survival and growth of Cl. botulinum , peeled, vacuum-packed potatoes for storage at ambient temperature should be given a heat treatment equivalent to an F₀3 process. If they are not given such a heat treatment they should be stored at a temperature below 4°C.     

Evaluation of a monoclonal antibody-based immunoassay for detecting type B Clostridium botulinum toxin produced in pure culture and an inoculated model cured meat system

A monoclonal antibody-based amplified ELISA method for detecting Clostridium botulinum type B toxin was evaluated for its ability to detect the toxin in the supernatant fluid of pure cultures and after growth from Cl. botulinum spores inoculated into pork slurries. Slurries containing NaCl (1.5–4.5%w/v) and polyphosphate (0.3%w/v) were either unheated or heated 80°C/5 min followed by 70°C/2 h before incubation at 15°, 20° or 27°C. Presence of specific toxin was confirmed by mouse bioassay and results were compared with those of the amplified ELISA method. A total of 48 strains, consisting of 38 Cl. botulinum and 10 Cl. sporogenes (putrefactive anaerobes), and 140 slurry samples were tested. Cultures of eight out of nine strains of type B Cl. botulinum and 73 of 101 slurry samples containing type B toxin were positive by ELISA; the remaining 28 slurry samples contained type B toxin at levels below or close to the detection limit (20 LD₅₀/ml) of the type B ELISA. No falsepositive reactions occurred with Cl. botulinum types A, C, D, E or F, or with the 10 strains of Cl. sporogenes. Toxin produced by one strain of Cl. botulinum type B (NCTC 3807) was not detected by this single monoclonal antibody-based amplified ELISA. With a mixture of two monoclonal antibodies, however, the toxin from NCTC 3807 could be detected without reducing the sensitivity of the ELISA.     

Combined effect of water activity and pH on inhibition of toxin production by Clostridium botulinum in cooked, vacuum-packed potatoes.

The effects of water activity (aw, 0.955 to 0.970), pH (4.75 to 5.75), and storage time (up to 60 days) on toxin production by Clostridium botulinum in cooked, vacuum-packed potatoes were studied by using factorial design experiments and most-probable-number methodology. Samples were inoculated with 10(3), 10(4), or 10(5) spores of a mixture of five type A and five proteolytic type B strains, incubated at 25 degrees C, and analyzed for toxin production. Toxin was produced at pH levels of greater than or equal to 4.75 when the aw was greater than or equal to 0.970, pH greater than 5.25 when the aw was 0.965, and pH greater than or equal to 5.75 at an aw of 0.960. No toxin was detected when the aw was 0.955. The probability of toxigenesis was significantly affected (P less than 0.0001) by storage time, aw, pH, and the interactions aw.pH and aw.storage time. The response to a decrease in pH was linear, while the response to a decrease in aw was curvilinear. Using multiple linear regression, equations were derived which could predict the length of time until toxin production and the probability of toxigenesis by a single spore under defined conditions.     

Combined effect of water activity and pH on inhibition of toxin production by Clostridium botulinum in cooked, vacuum-packed potatoes

The effects of water activity (aw, 0.955 to 0.970), pH (4.75 to 5.75), and storage time (up to 60 days) on toxin production by Clostridium botulinum in cooked, vacuum-packed potatoes were studied by using factorial design experiments and most-probable-number methodology. Samples were inoculated with 10(3), 10(4), or 10(5) spores of a mixture of five type A and five proteolytic type B strains, incubated at 25 degrees C, and analyzed for toxin production. Toxin was produced at pH levels of greater than or equal to 4.75 when the aw was greater than or equal to 0.970, pH greater than 5.25 when the aw was 0.965, and pH greater than or equal to 5.75 at an aw of 0.960. No toxin was detected when the aw was 0.955. The probability of toxigenesis was significantly affected (P less than 0.0001) by storage time, aw, pH, and the interactions aw.pH and aw.storage time. The response to a decrease in pH was linear, while the response to a decrease in aw was curvilinear. Using multiple linear regression, equations were derived which could predict the length of time until toxin production and the probability of toxigenesis by a single spore under defined conditions.     

Growth of and toxin production by nonproteolytic Clostridium botulinum in cooked puréed vegetables at refrigeration temperatures.

Seven strains of nonproteolytic Clostridium botulinum (types B, E, and F) were each inoculated into a range of anaerobic cooked puréed vegetables. After incubation at 10 degrees C for 15 to 60 days, all seven strains formed toxin in mushrooms, five did so in broccoli, four did so in cauliflower, three did so in asparagus, and one did so in kale. Growth kinetics of nonproteolytic C. botulinum type B in cooked mushrooms, cauliflower, and potatoes were determined at 16, 10, 8, and 5 degrees C. Growth and toxin production occurred in cooked cauliflower and mushrooms at all temperatures and in potatoes at 16 and 8 degrees C. The C. botulinum neurotoxin was detected within 3 to 5 days at 16 degrees C, 11 to 13 days at 10 degrees C, 10 to 34 days at 8 degrees C, and 17 to 20 days at 5 degrees C.     

Survival of Campylobacter jejuni on beef trimmings during freezing and frozen storage

AIMS: To investigate the survival of two animal isolates of Campylobacter jejuni on beef trimmings during freezing and frozen storage. METHODS AND RESULTS: Meat packs inoculated with 10(3) or 10(6) cfu g(-1) of either strain of C. jejuni were frozen to -18 degrees C, and sampled at regular intervals over 112 d storage to determine Campylobacter numbers and sublethal injury. For both strains and inoculation levels the numbers of Campylobacter decreased in the first 7 d of storage by ca. 0.6-2.2 log cfu g(-1) and then remaining constant over the remainder of the storage trial, with neither isolate exhibiting sublethal injury. CONCLUSIONS: Despite an initially significant decrease in number, these pathogens were able to survive standard freezing conditions in meat, but did not exhibit sublethal injury. SIGNIFICANCE AND IMPACT OF THE STUDY: Strict hygiene and/or the implementation of decontamination technologies are recommended as a means to assure the safety of meat with respect to this pathogen.     

Isolation and properties of highly purified C1. botulinum toxin type E]

A new method of isolation of highly purified Cl. botulinum toxin of E type from the cultural fluid of strain 188 centrifugates was developed. The method allows to isolate the toxin both in a precursor and in activated forms with a yield of 10--15%. The method includes fractionation by ammonium sulfate, ultrafiltration and subsequent column chromatography on DEAE-cellulose, Sephadex G-200 and DEAE-Sephadex A-50. The preparations were found homogeneous during polyacrylamide gel electrophoresis and immunoprecipitation in agar with antitoxic horse serum. The potential specific toxicity of the preparations is 1--1,2.10(7) DLM/mg of protein. The molecular weight of the toxin is about 160 000; the molar extinction coefficient is equal to 278 nm. The isoelectric point lies around pH 6.0. The highly purified Cl. botulinum toxin of E type was found stable upon storage.     

Effect of Nitrite and Nitrate on Toxin Production by Clostridium botulinum and on Nitrosamine Formation in Perishable Canned Comminuted Cured Meat

Comminuted ham was formulated with different levels of sodium nitrite and nitrate, inoculated with Clostridium botulinum, and pasteurized to an internal temperature of 68.5 C. When added to the meat, nitrite concentrations decreased, and cooking had little effect on them. Nitrite concentrations decreased more rapidly during storage at 27 than at 7 C; however they remained rather constant at formulated levels throughout the experiment at both incubation temperatures. The level of nitrite added to the meat greatly influenced growth and toxin production of C. botulinum. The concentration of nitrite necessary to effect complete inhibition was dependent on the inoculum level. With 90 C. botulinum spores/g of meat, botulinum toxin developed in samples formulated with 150 but not with 200 mug of nitrite per g of meat. At a spore level of 5,000/g, toxin was detected in samples with 400 but not with 500 mug of nitrite per g of the product incubated at 27 C. At lower concentrations of nitrite, growth was retarded at both spore levels. No toxin developed in samples incubated at 7 C. Nitrate showed a statistically significant inhibitory effect at a given nitrite level; however, the effect was insufficient to be of practical value. Analyses for 14 volatile nitrosamines from samples made with varying levels of nitrite and nitrate were negative at a detection level of 0.01 mug of nitrite or nitrate per g of meat.     

The Growth and Toxin Production of Clostridium botulinum Type E in Certain Vacuum Packed Fish

The growth and toxin production of Clostridium botulinum type E in various types of vacuum packed fish products was tested, with particular reference to the time/temperature relationship of storage. Toxin production was most rapid in herring although smoking retarded its development. With as small an inoculum as 10² spores/pack, fresh herring became toxic after storage for 15 days at 5°. Irradiation of three species of fish after inoculation with Cl. botulinum type E showed that spores surviving radiation germinated and produced toxin more rapidly than an equivalent concentration of spores in nonirradiated fish.     

Growth and toxin production by non-proteolytic and proteolytic Clostridium botulinum in cooked vegetables

Growth and toxin production by proteolytic and non-proteolytic strains of Clostridium botulinum have been followed in 28 cooked puréed vegetables prepared under strict anaerobic conditions and incubated at 30°C for up to 60 d. Toxin production was confirmed in 25 of the cooked vegetables inoculated with a suspension of spores of proteolytic strains of types A and B, and in 13 inoculated with a suspension of spores of non-proteolytic strains of types B, E and F. For both proteolytic and non-proteolytic strains, a trend was identified correlating growth and toxin production with the pH of the cooked puréed vegetables.     

Evaluation of the use of the bioMerieux Rapid ID32 A for the identification of Clostridium botulinum

The neurotoxins produced by Clostridium botulinum are amongst the most potent known to man. Toxin production is detected by a mouse bioassay, which requires several days for a result and is not acceptable for routine use unless there is a high level of suspicion. The Rapid ID32 A kit produced by bioMerieux gives an identification of an isolate within 4 h. The aim of this study was to examine the efficiency of the identification of Cl. botulinum using the Rapid ID32 A. Forty-two strains of Cl. botulinum , one strain each of botulinum toxin-producing Cl. butyricum and Cl. baratii , and four strains of Cl. sporogenes , were tested. One strain of Group I Cl. botulinum gave a presumptive identification of Group II Cl. botulinum , six strains of Cl. botulinum were identified as 50–98% Cl. botulinum in some tests, and 17 strains of Cl. botulinum were identified as <50% Cl. botulinum. Thirteen strains of Cl. botulinum were identified as >99% Cl. sporogenes or 86% Cl. histolyticum , and five strains gave a combination of these results. All strains of Cl. sporogenes were correctly identified. These results show that some strains of Cl. botulinum may not be correctly identified using the Rapid ID32 A.